KR20050023045A - Gamma-Butyrolactone autoregulator receptor gene(EsgR) isolated from Saccharopolyspora erythraea and protein expressed thereof - Google Patents

Abstract

PURPOSE: A gamma-Butyrolactone autoregulator receptor gene(EsgR) isolated from Saccharopolyspora erythraea and a protein expressed therefrom are provided, which gene and protein are useful for studies on induction of erythromycin production, induction of second metabolism production, and spore formation using Saccharopolyspora sp. CONSTITUTION: The gamma-Butyrolactone autoregulator receptor gene(EsgR) isolated from Saccharopolyspora erythraea IFO 13426 has the nucleotide sequence set forth in SEQ ID NO:1. The protein encoded by the gamma-Butyrolactone autoregulator receptor gene(EsgR) isolated from Saccharopolyspora erythraea IFO 13426 of SEQ ID NO:1 has the amino acid sequence set forth in SEQ ID NO:2.

Description

Gamma-Butyrolactone autoregulator receptor gene (EsgR) isolated from Saccharopolyspora erythraea and protein expressed codes} isolated from Saccharopolyphora erythrrea

The present invention relates to a γ-butyrolactone self-regulatory receptor gene EsgR isolated from Saccharopolyspora erythraea IFO 13426 and a protein expressed therefrom.

Actinomycetes are Gram-positive bacteria that are unique microorganisms that are largely separated from soil. They perform remarkable morphology like filamentous fungi and have a life cycle of basal mycelia, aerial mycelium, conidia and spore germination. In addition to this morphology, it produces industrially useful secondary metabolites such as antibiotics, bioactive substances, and pigments.

The secondary metabolites produced by actinomycetes occur in the late stage of culture, which is the period of sporulation, indicating that the production of secondary metabolites is closely related to morphological differentiation. This morphological differentiation and physiological differentiation of secondary metabolite production are small molecule signaling agents called autoregulators produced by actinomycetes and self-regulatory receptor proteins that specifically bind to them. It is known that by the interaction of. These factors are pleiotropic and many studies have been conducted on Streptomyces genus actinomycetes. The self-regulatory factors so far identified are considered to be prokaryotic hormones as they correspond to hormones of higher organisms because they act at very low concentrations of several ng / ml and induce secondary metabolism or form differentiation.

Γ-butyrolactone such as A-factor, factor I, virginia butanolides, VB-A, B, C, D, and E Ten different self-regulatory factors with rings have been isolated and their structure is revealed, and their function is being studied at the molecular level.

According to Waki et al., It is estimated that more than 60% of Streptomyces Actinomycetes produce self-regulatory or similar self-regulatory agents so far, and specific hormone proteins are present in hormones of higher organisms. , Streptomyces (Streptomyces) in the self-control factor actinomycetes were also estimated harirago the specific receptor protein present. Indeed, starting with BarA [18] from S. virginiae , ArpA [14] from S. griseus , Streptomyces Ravendulae From FRI-5, FarA was isolated as a specific receptor protein of self-regulatory factor and the genes were cloned.

Among the self-regulatory and receptor proteins that have been identified to date, virginiae butanolides (VBs) and VB receptor proteins (BarA) from S. virginiae are the most studied. VB A-E was isolated from S. virginiae and its structure was determined. As a part of the study on the mechanism of antibiotic biosynthesis in relation to signal transduction of these inducers, Kim et al. Among the VBs, VB-C receptor, a binding protein involved in the signal transduction of VB-C, was first identified and purified. Also, in vivo and In the in vitro experiment, in the absence of VB, BarA binds to a specific DNA sequence of the target gene or operon promoter region and inhibits transcription. When VB is produced, it binds to BarA, which is bound to DNA. BarA was identified as a DNA-binding transcription inhibitor in the result of initiating transcription of the target gene or operon from the promoter region, and the VB-BarA system is composed of virginiamycin M1 (VM1) and virgininycin, which differ in structure. (virginiamycin) S (VS) production has been shown to co-regulate. In addition, according to Lee et al., The barB -varS operon is located downstream of the barA gene, and the varS gene is Viriniamycin.

mycin), a VS-specific efflux protein involved in resistance.

As an application of these inducers, Streptomyces longudensis, a lysocellin-producing bacterium by Kim et al.S. longwoodensisInduction capacity of lysoceline and other antibiotics and pigments by VB-CS. erythraeus) Induction of erythromycin (EM) production by VB-C has been suggested from IFO 13426.

Saccharide in the form of a Streptomyces used in the present invention Indianapolis Fora Erie waste ah (Saccharo-

Erythromycin produced by polyspora erythraea) is a bacteriostatic drug that is more permeable to the cell wall of gram-positive bacteria than gram-negative bacteria, and has 100 times more activity on gram-positive bacteria than three gram-negative bacteria. It is used in the majority of infections caused by bakteo (Campylobacter) enterocolitis, neonatal conjunctivitis, Legionella disease (Legionnarire 's disease), preoperative preparation, mycoplasma (Mycoplasma), chlamydia (Chlamydia) infections and gram positive bacteria. The antimicrobial activity is largely similar to penicillin G and is an alternative to penicillin hypersensitivity, especially in infections caused by streptococci, staphylococci, pneumococci, and Clostridia .

Accordingly, an object of the present invention is to identify gamma-butyrolacton self-regulatory receptor proteins and genes encoding them that are involved in Saccharopolyspora erythraea IFO 13426 secondary metabolic regulation. It is to prepare a platform to identify the production control mechanism of erythromycin.

The present invention relates to a γ-butyrolactone self-regulatory receptor gene EsgR isolated from Saccharopolyspora erythraea IFO 13426 and a protein expressed therefrom.

The technical problem of the present invention is gamma-butyrolactone of Saccharopolyspora erythraea IFO 13426 by PCR using a common array of the Streptomyces gene gene as a primer. ) Was achieved by cloning the autoregulator receptor gene.

As described above, although a large number of receptor genes of the self-regulatory gene of Streptomyces genus have been found to date, the receptor gene of the self-regulatory factor of Saccharopolyspora has not been identified. Receptor genes of self-regulatory genes in the genus Saccharopolyspora were first identified, enabling the study of induction of antibiotics such as erythromycin, induction of secondary metabolites and sporulation using Saccharopolyspora .

The present invention relates to a gamma-butyrolactone self-regulatory receptor gene EsgR isolated from Saccharopolyspora erythraea IFO 13426 and a protein expressed therefrom.

Strains and plasmids used in the Examples for the present invention are as follows. As a strain for cloning the receptor gene, Actinomycetes Saccharopolyspora erythraea IFO 13426 was used. Cloning vector was used for pUC19, and the plasmid transfected cells were E. coli DH5 ( supE 44 l ac U169 (80 lacZ M15) hsdR 17 recA 1 endA 1 gyrA 96 thi -1 relA 1) coli (E. coli) XL10-Gold ( Tet R (mcrA) 183 (mcrCB - hsdSMR - mrr) 173 endA 1 supE 44 thi -1 recA 1 gyrA 96 relA 1 lac Hte [F 'proAB, laclqZ M15 Tn 10 (Tet R) Amy Cam R]).

The medium and conditions used in the present invention are as follows.

The culture of the test bacteria was performed using ISP medium No. Slant culture was carried out for 3 weeks at 28 ℃ using 2, liquid culture for the extraction of chromosomal DNA was 50 ml (250 ml Erlenmeyer flasks) tryptic Soy Broth of platinum from the slant , TSB) medium was incubated for 48 hours at 28 ℃, 200 rpm, the main culture was carried out for 4 days inoculated with 100% (500 ml baffled flask TSB medium 3% pre-cultured). E. coli was incubated for 18 hours at 37 ° C. with ampicillin (50 μg / ml) in Luria Bertani (LB) medium and all reagents used were Difco Laboratories (Difco Laboratories). Laboratories Co, (USA)) and Sigma Chemical Co. USA.

Example 1 Saccharopolis Fora Eritrea Saccharopolyspora erythraea Probe Probe to Receptor Gene from IFO 13426

First, isolation of Saccharopolyspora erythraea IFO 13426 genome and plasmid DNA is isolated. The genomic DNA isolation of the bacteria was carried out according to the Hopwood method, and the plasmid DNA was isolated from the Flexi Prep Kit (Amersham Pharmacia Biotech., UK), the TELT method, and Alkaline-SDS. The extraction was carried out.

1: 1 binding and dimer formation, which is a common feature of self-regulatory receptor proteins studied previously to confirm the presence of receptor genes from Saccharopolyspora erythraea The helix-turn-helix motif of the helix-turn-helix motif estimated at the N-terminal region (see FIG. 1) is high at both ends. After constructing a specific forward primer (AF-V, 5'-CGCGGATCCGCSGCSGCSNNNGTSTTCGA-3 ') and a reverse primer (AR-1, 5'-CGCGGATCCGAAGTGGAAGTASAGSGCSCC-3') including a Bam HI site, the chromosomal DNA of the test specimen (100) ng / ml) as template, 10 pmole primer, 2.5 mM dNTPs, 1/8 dil. Polymerase chain reaction (hereinafter referred to as PCR) was performed with the composition of Ex Taq , 10X Ex Taq buffer. PCR reaction was amplified 35 times for 5 min at 95 ℃, 1 min at 95 ℃, 40 sec at 66 ℃, 50 sec at 72 ℃ and stored at 4 ℃.

The PCR product obtained by the above method was electrophoresed using 2% agarose horizontal gel, and TAE (40 mM tris-acetate, pH 8.0, 1 mM EDTA) was used as the buffer solution. A 120 bp PCR product was identified (see Figure 2. A).

The DNA fragments were recovered by lyolysis, treated with Bam HI, linked to pUC19, and transformed into E. coli DH5. The transforming bacteria were incubated at 37 ° C. for 18 hours and plated with transforming bacteria in LB solid medium (0.4% X-gal, 0.1 M IPTG smear) to which ampicillin (ampicillin, 50 μg / ml) was added. (colony) was selected as transforming bacteria. Competent cells were prepared by the Hanahan method, and all restriction enzymes, modifiers, and markers were used by Takara.

Selected white colony was inoculated in 3 ml LB liquid medium to which ampicillin (50 μg / ml) was added and then incubated at 37 ° C. for 18 hours. Plasmids were isolated from Flexi Prep Kit from three (R-1, R-2, and R-3) selected transformants and electrophoresed on 1% agarose gel by Bam HI treatment. In addition, pUC19 (2.7 kbp) in addition to the PCR products at each 120bp position PCR fragments of Saccharopolyspora erythraea was confirmed that the E. coli DH5 was transformed (Fig. 2. B Only plasmids with confirmed transformation were used for sequencing. Plasmids used in sequencing were separated by alkaline-SDS extraction.

Transformed plasmids were tested with Thermo Sequenase fluorescent labeledled primer cycle sequencing kit with 7-deaza-dGTP, Amersham Pharmacia Biotech.UK. PCR was performed using Cy5-labeled M13 primer, and the nucleotide sequence was determined using a fluorescence DNA sequencer (ALFred, Amersham Pharmacia Biotech.UK). Sequencing and homology comparisons were performed using a Genetics-Win 3.2 version (GENETYX-WIN Ver 3.2, Software Develoment Co., Japan) and a DNA database. As a result, Fig. As shown in Fig. 3, homology with the receptor gene (R-gene) derived from the known Streptomyces genus was confirmed in the AF-V and AR-1 regions of the primers used, and the preservation of the primer portion (FIG. 1. High). These results suggest that PCR method targeting receptor genes is a very effective means to search for receptors.

Plasmids identified as part of the receptor gene were treated with Bam HI to recover the PCR product by freezing and then radioisotope with a random primer DNA Labeling Kit version 2 (Takara Shuzo Co., Japan). Probes were prepared for Southern blot and colony hybridization by labeling with the element [-32P] dCTP.

Example 2 pESG Screening via Southern blot Analysis and Colorny Hybridization

U Fora Erie waste Ah 10 kinds of restriction enzymes, located on (hereinafter referred to as the Multi Cloning Site, MCS) (Saccharopolyspora erythraea) IFO 13426 pUC19 multi-cloning site of the chromosomal DNA of a derived saccharide (Bam HⅠ, Eco RⅠ, Hind Ⅲ, Kpn Ⅰ, Pst I, Sac I, Sal I, Sma I, Sph I, Xba I), respectively, followed by electrophoresis on 1% agarose gel and agarose gel modified solution (0.5 M sodium hydroxide, 1.5 M sodium chloride). ) Was treated once every 30 minutes, and then treated with neutralizing solution (1.5 M sodium chloride, 0.5 M tris-hydrochloric acid, 1 mM EDTA, pH 7.2) twice for 15 minutes. After that, the DNA of the agarose gel was transferred to a nylon membrane (Hybond-N +, Amersham Pharmacia Biotech., UK), and the DNA was fixed by UV irradiation for 5 minutes.

20 ml of a fast hybrid buffer (Amersham Pharmacia Biotech.UK) and a membrane were placed in a hybrid bag, prehybridized for 15 minutes, and then labeled with a radioisotope [-32P] dCTP probe. Was added and hybridized at 65 ° C. for 2 hours. To remove nonspecific signals of the hybridized membranes, they were washed under conditions of 2 × SSC / 0.1% SDS (rt), 1 × SSC / 0.1% SDS (rt), 0.2 × SSC / 0.1% SDS (65). It was then exposed to an X-ray film (X-ray film) at -80 ℃ for 1 hour and then developed.

Southern blot showed restriction enzymeKpnⅠ,PstⅠ,SacⅠ,SalⅠ,SmaⅠ,SphStrong positive signals were seen at various locations in I (see FIG. 5). Of these positive signals, about 3.2 kbpSacI fragment (Esg) Was confirmed as the target DNA fragment for cloning the receptor protein gene of the test organism.

The chromosomal DNA of the test bacteria was treated with restriction enzyme Sac I, electrophoresed in 1% agarose gel, and a DNA fragment of about 3.2 kbp was recovered and linked to the Sac I site of pUC19. E. coli XL10-Gold, a competent cell, was transformed to prepare a DNA library of about 1000 3.2 kbp Sac I fragments. The color needles were transferred to nylon membrane, and then treated with 10% SDS, denatured solution, neutralized solution, and 2 × SSC solution at room temperature for 5 minutes, and the DNA was fixed by UV irradiation for 5 minutes. Probe and subsequent work used for Colony hybridization were performed in the same manner as Southern hybridization.

Colorney hybridization resulted in the strongest positive signal in one of the 1000 color teeth, and the plasmid containing the Sac I fragment of 3.2 kbp was named pESG.

Example 3. 2 kbp Sac I Determination of EsgR by Subcloning and Sequencing of Fragments

In order to determine all the nucleotide sequences of 3.2 kbp DNA fragments obtained through colorney hybridization, the restriction enzymes contained in the MCS of pUC19 were subjected to electrophoresis on 1% agarose gel, whereby restriction enzyme sites could be located. Restriction maps were prepared by subcloning using only portions. In this case, pUC19 was used as a vector and competent cells were used as E. coli DH5, and DNA sequencing was performed in the same manner as previously described. Based on the restriction map, Sal I was treated to recover 1.4 kbp, 1.1 kbp and 0.7 kbp sized DNA fragments, then subcloned and sequenced and analyzed. As a result, some of the self-regulatory receptor protein genes were 0.7 kbp. The possibility of being present at the terminal end of the DNA fragment was suggested.

As a result of the subcloning of pESG, only part of the receptor gene was identified at 0.7 kbp, so that the full sequencing of pESG was performed to determine the position and size of the receptor gene. Base alignment was determined by primer walking using M13 forward and reverse primers. To an area in which the gene encoding the self-control factor receptor protein gene as shown in the result of repeatedly sequenced three times to ensure the accuracy of the nucleotide sequence Figure 6 contains a Kpn Ⅰ and Sal Ⅰ of the Esg fragment contained in pESG It can be seen that it exists, it was named Esg R. Known receptor proteins contain 232 BarAs, 276 ArpAs, 221 FarAs, 215 ScbRs, and EsgR, a Saccharopolyspora erythraea IFO 13426, consists of 205 amino acids. It was confirmed (FIG. 7).

In order to efficiently determine the nucleotide sequence of Sac I 3.2 kbp DNA fragment ( Esg ) derived from Saccha. Erythraea , the restriction enzyme located in the MCS of pUC19 was subjected to electrophoresis, followed by electrophoresis. Restriction maps were prepared by subcloning using only those parts that can be located. Based on this restriction map, Sal I was treated to recover 1.4 kbp, 1.1 kbp, 0.7 kbp sized DNA fragments, and then subjected to subcloning, sequencing and analysis. As a result, a portion of the self-regulatory receptor protein gene was 0.7 kbp. The possibility of being present at the end of the fragment has been suggested.

The nucleotide sequence of the EsgR gene was tested for homology using the BLAST and Genetics-Win 3.2 version (GENETYX-Win Ver. 3.2) computer programs of the DNA database. As a result, as shown in Table 1 and FIG. 8, the homologous genes of the self-regulatory receptor protein genes derived from Streptomyces sp. Showed at least 30% homology at the amino acid level.

EsgR ( S.erythraea ) sameness(%) positivity(%) FarA ( S. lavendulae ) 34 47 ScbR ( S. coelicolor A3 (2)) 32 47 ArpA ( S.griseus ) 31 46 BarA ( S.virginiae ) 32 45

In addition, EsgR codes for the helix-turn-helix DNA binding motif (FIG. 9) that existing self-regulatory receptor proteins possess for controlling antibiotic biosynthesis genes below, and also used primers AR-1 and EsgR is self-regulated by Saccha. Erythraea in that the AF-V region is not completely consistent with conventional receptors (FIGS. 1, 3, 9) but shows a fairly high homology . It was assumed to be a gene encoding factor receptor proteins. Currently, the presence of regulatory genes in the upstream and downstream regions of receptor genes is being searched for, and further studies on erythromycin production are under way.

The present invention relates to a gamma-butyrolactone self-regulatory receptor gene (EsgR) isolated from Saccharopolyspora erythraea IFO 13426 and a protein expressed therefrom. As described above, a large number of receptor genes of the self-regulatory gene of Streptomyces genus have been found, but no receptor genes of the self-regulatory factor of the genus Saccharopolyspora have been identified. Receptor genes of the self-regulatory factor of Saccharo-polyspora were first identified, enabling the use of Saccharopolyspora to induce the production of antibiotics such as erythromycin, induce the production of secondary metabolites and spore formation. The invention is in the biotechnology and pharmaceutical industries It is an excellent invention.

1 is a diagram showing a method of designing a PCR primer for cloning a gene encoding a gamma-butyrolactone self-regulatory receptor protein and a gene sequence of the primer.

FIG. 2 shows DNA fragments obtained by PCR using the primers of FIG. 1 as templates for genes and plasmids derived from Saccharopolyspora erythraea IFO 13426 and transformed E. coli DH5. The photo shows the results of electrophoresis.

3 is a diagram comparing the amino acid sequence of the PCR product obtained from Saccharopolyspora erythraea IFO 13426 with the amino acid sequences of known self-regulatory receptor proteins.

Figure 4 shows a 120 bp probe containing a portion of the receptor gene obtained from Saccharopolyspora erythraea IFO 13426.

Figure 5 is a photograph showing the results of Southern blot analysis of the gene of Saccharopolyspora erythraea IFO 13426.

6 is a diagram showing the position of a gene encoding a self-regulatory receptor protein.

7 shows the nucleotide sequence of the EsgR gene.

8 is a diagram showing the amino acid sequence and the upstream nucleic acid sequence of the protein encoded by EsgR .

9 is a diagram comparing the amino acid sequence of EsgR and BarA .

10 is a diagram showing the sequence of the amino-terminal of the EsgR -like proteins obtained from the database.

<110> KIM, hyunsu <120> Gamma-butyrolactone autoregulator receptor gene isolated from Saccharopolyspora erythraea and protein EsgR expressde <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 615 <212> DNA <213> Saccharopolyspora erythraea <400> 1 atgccccagc agcgtcgggc gcaagccact cggcgaacga tcctggtcgc ggccgcggag 60 gagttcgacc gcgtcggcta cgaggcgacc acgctcaacg gcatcctgcg tcgcggcggg 120 gtcaccaagg gcgccttcta cttccacttc gcgtccaagg aagacgtcgc gcgggtgctc 180 gtccgggagc agcgcgaacg ctggcccgcg ctctggcggc gctgggtccg ccggggtctc 240 gacccgttgt ccacggcgat cggcctggtc gacgagctgc tcgcggtgct cgacgaggac 300 gtggcgacgc gggccgggat gcgactggcc tgccggcgcg agatcgaggc cgccgacccg 360 ccggactggg agcgggtcct gaccgagctg ctcgaccgcg ccgctgaccg cgggtacctc 420 ctgccgacgg tcgatccccg cgcgctcgcg cgggtggtgt actcggcgct tgtaggcgct 480 cggacgatcg atcgcggcgc ggtgtccacc gggcgcaccg ccgaggtgtg gcggatcgtc 540 ctgcgcggcg ccgcgacccc ggagtggctt cgcaccaacc cggtgctcca gccgcccgac 600 gaacgccccc gctga 615 <210> 2 <211> 204 <212> PRT <213> Saccharopolyspora erythraea <400> 2 Met Pro Gln Gln Arg Arg Ala Gln Ala Thr Arg Arg Thr Ile Leu Val 1 5 10 15 Ala Ala Ala Glu Glu Phe Asp Arg Val Gly Tyr Glu Ala Thr Thr Leu 20 25 30 Asn Gly Ile Leu Arg Arg Gly Gly Val Thr Lys Gly Ala Phe Tyr Phe 35 40 45 His Phe Ala Ser Lys Glu Asp Val Ala Arg Val Leu Val Arg Glu Gln 50 55 60 Arg Glu Arg Trp Pro Ala Leu Trp Arg Arg Trp Val Arg Arg Gly Leu 65 70 75 80 Asp Pro Leu Ser Thr Ala Ile Gly Leu Val Asp Glu Leu Leu Ala Val 85 90 95 Leu Asp Glu Asp Val Ala Thr Arg Ala Gly Met Arg Leu Ala Cys Arg 100 105 110 Arg Glu Ile Glu Ala Ala Asp Pro Pro Asp Trp Glu Arg Val Leu Thr 115 120 125 Glu Leu Leu Asp Arg Ala Ala Asp Arg Gly Tyr Leu Leu Pro Thr Val 130 135 140 Asp Pro Arg Ala Leu Ala Arg Val Val Tyr Ser Ala Leu Val Gly Ala 145 150 155 160 Arg Thr Ile Asp Arg Gly Ala Val Ser Thr Gly Arg Thr Ala Glu Val 165 170 175 Trp Arg Ile Val Leu Arg Gly Ala Ala Thr Pro Glu Trp Leu Arg Thr 180 185 190 Asn Pro Val Leu Gln Pro Pro Asp Glu Arg Pro Arg 195 200

Claims (2)

Esg R., a gene of gamma-butyrolactone self-regulatory receptor protein, having the nucleotide sequence of SEQ ID NO: 1, isolated from Saccharopolyspora erythraea IFO 13426 . A gamma-butyrolactone self-regulatory receptor protein having a sequence of SEQ ID NO: 2 isolated from Saccharopolyspora erythraea IFO 13426 encoded by the gene Esg R of claim 1.